Polypeptides having phospholipase C activity and polynucleotides encoding same

ABSTRACT

The present invention relates to isolated polypeptides having phospholipase C activity, and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 16/152,043filed Oct. 4, 2018, which a divisional of U.S. application Ser. No.14/649,084 filed Jun. 2, 2015, now abandoned, which is a 35 U.S.C. 371national application of international application no. PCT/CN2013/089106filed Dec. 11, 2013, which claims priority or the benefit under 35U.S.C. 119 of international application no. PCT/CN2012/086378 filed Dec.11, 2012 and U.S. provisional application No. 61/756,745 filed Jan. 25,2013. The content of these applications is fully incorporated herein byreference.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to polypeptides having phospholipase Cactivity and polynucleotides encoding the polypeptides. The inventionalso relates to nucleic acid constructs, vectors, and host cellscomprising the polynucleotides as well as methods of producing and usingthe polypeptides.

Further, the present invention relates to a method for reducing thecontent of phosphorous containing components in edible oil comprising ahigh amount of non-hydratable phosphorus, by the use of the enzymehaving phospholipase C activity.

Description of the Related Art

Several types of phospholipases are known which differ in theirspecificity according to the position of the bond attacked in thephospholipid molecule. Phospholipase A1 (PLA1) removes the 1-positionfatty acid to produce free fatty acid and 1-lyso-2-acylphospholipid.Phospholipase A2 (PLA2) removes the 2-position fatty acid to producefree fatty acid and 1-acyl-2-lysophospholipid. The term phospholipase B(PLB) is used for phospholipases having both A1 and A2 activity.Phospholipase C (PLC) removes the phosphate moiety to produce 1,2diacylglycerol and phosphate ester. Phospholipase D (PLD) produces1,2-diacylglycero-phosphate and base group.

Polypeptides having phospholipase C activity may be applied in anindustrial process, e.g., for refining of vegetable oils. Beforeconsumption vegetable oils are degummed to provide refined storagestable vegetable oils of neutral taste and light color. The degummingprocess comprises removing the phospholipid components (the gum) fromthe triglyceride rich oil fraction.

Traditionally, the degumming process has been based on either waterextraction, acidic or caustic treatment followed by a separationprocess. Due to the emulsifying properties of the phospholipidcomponents, the degumming procedure has resulted in a loss of oil i.e.of triglycerides.

Enzymatic degumming reduces the oils loss due to an efficient hydrolysisof the phospholipids which decrease the emulsifying properties. There isa need for further enzymes having phospholipase C activity and suitablefor application in enzymatic degumming of edible oils.

An enzyme having phospholipase C activity and 88% sequence identity withthe enzyme of the present invention is known from WO 2012062817.

SUMMARY OF THE INVENTION

The inventors have isolated a new enzyme having phospholipase C activityfrom a strain of Penicillium, or more specifically from a strain ofPenicillium emersonii found 2007 in China. Accordingly, the presentinvention provides novel polypeptides having phospholipase C activityand polynucleotides encoding the polypeptides.

The present invention relates to isolated polypeptides havingphospholipase C activity selected from the group consisting of:

(a) a polypeptide having at least 90% sequence identity to the maturepolypeptide of SEQ ID NO: 2;

(b) a polypeptide encoded by a polynucleotide that hybridizes under veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 1, or (ii) the full-length complement of (i);

(c) a polypeptide encoded by a polynucleotide having at least 90%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1;

(d) a variant of the mature polypeptide of SEQ ID NO: 2 comprising asubstitution, deletion, and/or insertion at one or more (e.g., several)positions; and

(e) a fragment of the polypeptide of (a), (b), (c), or (d) that hasphospholipase C activity.

The present invention also relates to isolated polynucleotides encodingthe polypeptides of the present invention; nucleic acid constructs;recombinant expression vectors; recombinant host cells comprising thepolynucleotides; and methods of producing the polypeptides.

The present invention also relates to a composition comprising thepolypeptides of the present invention

The present invention also relates to use of the aforementionedpolypeptide or the aforementioned composition in a process forhydrolysis of phospholipids, such as in a process to reduce thephospholipid content in an edible oil.

The present invention also relates to a transgenic plant, plant part orplant cell transformed with a polynucleotide encoding the polypeptidesof the present invention.

Definitions

Phospholipase C activity: The term “phospholipase C activity” means theactivity that catalyzes the reaction: Aphosphatidylcholine+H₂O=1,2-sn-diacylglycerol+choline phosphate.Phospholipase C activity may be determined according to the proceduredescribed in “Materials and Methods”. An enzyme having “phospholipase Cactivity” may belong to EC 3.1.4.3.

The polypeptides of the present invention have at least 20%, e.g., atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, and at least 100% of the phospholipase Cactivity of the mature polypeptide of SEQ ID NO: 2.

The term “phospholipase C activity” means that a polypeptide havingphospholipase C will have activity towards one or more ofphosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidicacid (PA) and phosphatidyl inositol (PI).

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Catalytic domain: The term “catalytic domain” means the region of anenzyme containing the catalytic machinery of the enzyme.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic or prokaryotic cell. cDNA lacks intron sequences thatmay be present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA that is processed through a seriesof steps, including splicing, before appearing as mature spliced mRNA.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a polypeptide. Theboundaries of the coding sequence are generally determined by an openreading frame, which begins with a start codon such as ATG, GTG, or TTGand ends with a stop codon such as TAA, TAG, or TGA. The coding sequencemay be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding a maturepolypeptide of the present invention. Each control sequence may benative (i.e., from the same gene) or foreign (i.e., from a differentgene) to the polynucleotide encoding the polypeptide or native orforeign to each other. Such control sequences include, but are notlimited to, a leader, polyadenylation sequence, propeptide sequence,promoter, signal peptide sequence, and transcription terminator. At aminimum, the control sequences include a promoter, and transcriptionaland translational stop signals. The control sequences may be providedwith linkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe polynucleotide encoding a polypeptide.

Expression: The term “expression” includes any step involved in theproduction of a polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding apolypeptide and is operably linked to control sequences that provide forits expression.

Fragment: The term “fragment” means a polypeptide having one or more(e.g., several) amino acids absent from the amino and/or carboxylterminus of a mature polypeptide; wherein the fragment has phospholipaseC activity. In one aspect, a fragment contains at least 1700 amino acidresidues (e.g., amino acids 1 to 1700 of SEQ ID NO: 2), at least 1600amino acid residues (e.g., amino acids 1 to 1600 of SEQ ID NO: 2), or atleast 1500 amino acid residues (e.g., amino acids 1 to 1500 of SEQ IDNO: 2).

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Isolated: The term “isolated” means a substance in a form or environmentthat does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more or all of the naturally occurring constituents withwhich it is associated in nature; (3) any substance modified by the handof man relative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., multiple copiesof a gene encoding the substance; use of a stronger promoter than thepromoter naturally associated with the gene encoding the substance). Anisolated substance may be present in a fermentation broth sample.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. In one aspect, the maturepolypeptide is amino acids 1 to 594 of SEQ ID NO: 2. Amino acids −16 to−1 of SEQ ID NO: 2 are a signal peptide. It is known in the art that ahost cell may produce a mixture of two of more different maturepolypeptides (i.e., with a different C-terminal and/or N-terminal aminoacid) expressed by the same polynucleotide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving phospholipase C activity. In one aspect, the mature polypeptidecoding sequence is nucleotides 49 to 1830 of SEQ ID NO: 1. Nucleotides 1to 48 of SEQ ID NO: 1 encode a signal peptide.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic, which comprises one or more controlsequences.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used aregap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62(EMBOSS version of BLOSUM62) substitution matrix. The output of Needlelabeled “longest identity” (obtained using the −nobrief option) is usedas the percent identity and is calculated as follows:(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 5.0.0 or later. The parameters used are gap open penalty of 10,gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix. The output of Needle labeled “longestidentity” (obtained using the −nobrief option) is used as the percentidentity and is calculated as follows:(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Stringency Conditions:

Very low stringency conditions: The term “very low stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 45° C.

Low stringency conditions: The term “low stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 25% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at50° C.

Medium stringency conditions: The term “medium stringency conditions”means for probes of at least 100 nucleotides in length, prehybridizationand hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/mlsheared and denatured salmon sperm DNA, and 35% formamide, followingstandard Southern blotting procedures for 12 to 24 hours. The carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS at 55° C.

Medium-high stringency conditions: The term “medium-high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and either 35%formamide, following standard Southern blotting procedures for 12 to 24hours. The carrier material is finally washed three times each for 15minutes using 2×SSC, 0.2% SDS at 60° C.

High stringency conditions: The term “high stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 50% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at65° C.

Very high stringency conditions: The term “very high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 70° C.

Subsequence: The term “subsequence” means a polynucleotide having one ormore (e.g., several) nucleotides absent from the 5′ and/or 3′ end of amature polypeptide coding sequence; wherein the subsequence encodes afragment having phospholipase C activity. In one aspect, a subsequencecontains at least 1800 nucleotides (e.g., nucleotides 1 to 1800 of SEQID NO: 1), at least 1700 nucleotides (e.g., nucleotides 1 to 1700 of SEQID NO: 1), or at least 1600 nucleotides (e.g., nucleotides 1 to 1600 ofSEQ ID NO: 1).

Variant: The term “variant” means a polypeptide having phospholipase Cactivity comprising an alteration, i.e., a substitution, insertion,and/or deletion, at one or more (e.g., several) positions. Asubstitution means replacement of the amino acid occupying a positionwith a different amino acid; a deletion means removal of the amino acidoccupying a position; and an insertion means adding an amino acidadjacent to and immediately following the amino acid occupying aposition.

DETAILED DESCRIPTION OF THE INVENTION

Polypeptides Having Phospholipase C Activity

In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the mature polypeptide of SEQ ID NO: 2 ofat least 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100%, which have phospholipase C activity. In one aspect, thepolypeptides differ by no more than 10 amino acids, e.g., 1, 2, 3, 4, 5,6, 7, 8, or 9, from the mature polypeptide of SEQ ID NO: 2.

A polypeptide of the present invention preferably comprises or consistsof the amino acid sequence of SEQ ID NO: 2 or an allelic variantthereof; or is a fragment thereof having phospholipase C activity. Inanother aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 2. In another aspect, the polypeptidecomprises or consists of amino acids 1 to 594 of SEQ ID NO: 2.

In another embodiment, the present invention relates to an isolatedpolypeptide having phospholipase C activity encoded by a polynucleotidethat hybridizes under very low stringency conditions, low stringencyconditions, medium stringency conditions, medium-high stringencyconditions, high stringency conditions, or very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, or (ii) the full-length complement of (i) (Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor,N.Y.).

The polynucleotide of SEQ ID NO: 1 or a subsequence thereof, as well asthe polypeptide of SEQ ID NO: 2 or a fragment thereof, may be used todesign nucleic acid probes to identify and clone DNA encodingpolypeptides having phospholipase C activity from strains of differentgenera or species according to methods well known in the art. Inparticular, such probes can be used for hybridization with the genomicDNA or cDNA of a cell of interest, following standard Southern blottingprocedures, in order to identify and isolate the corresponding genetherein. Such probes can be considerably shorter than the entiresequence, but should be at least 15, e.g., at least 25, at least 35, orat least 70 nucleotides in length. Preferably, the nucleic acid probe isat least 100 nucleotides in length, e.g., at least 200 nucleotides, atleast 300 nucleotides, at least 400 nucleotides, at least 500nucleotides, at least 600 nucleotides, at least 700 nucleotides, atleast 800 nucleotides, or at least 900 nucleotides in length. Both DNAand RNA probes can be used. The probes are typically labeled fordetecting the corresponding gene (for example, with ³²P, ³H, ³⁵S,biotin, or avidin). Such probes are encompassed by the presentinvention.

A genomic DNA or cDNA library prepared from such other strains may bescreened for DNA that hybridizes with the probes described above andencodes a polypeptide having phospholipase C activity. Genomic or otherDNA from such other strains may be separated by agarose orpolyacrylamide gel electrophoresis, or other separation techniques. DNAfrom the libraries or the separated DNA may be transferred to andimmobilized on nitrocellulose or other suitable carrier material. Inorder to identify a clone or DNA that hybridizes with SEQ ID NO: 1 or asubsequence thereof, the carrier material is used in a Southern blot.

For purposes of the present invention, hybridization indicates that thepolynucleotide hybridizes to a labeled nucleic acid probe correspondingto (i) SEQ ID NO: 1; (ii) the mature polypeptide coding sequence of SEQID NO: 1; (iii) the full-length complement thereof; or (iv) asubsequence thereof; under very low to very high stringency conditions.Molecules to which the nucleic acid probe hybridizes under theseconditions can be detected using, for example, X-ray film or any otherdetection means known in the art.

In one aspect, the nucleic acid probe is nucleotides 55 to 1900nucleotides 100 to 1800, nucleotides 300 to 1200, or nucleotides 500 to1000 of SEQ ID NO: 1. In another aspect, the nucleic acid probe is apolynucleotide that encodes the polypeptide of SEQ ID NO: 2; the maturepolypeptide thereof; or a fragment thereof. In another aspect, thenucleic acid probe is SEQ ID NO: 1. In another embodiment, the presentinvention relates to an isolated polypeptide having phospholipase Cactivity encoded by a polynucleotide having a sequence identity to themature polypeptide coding sequence of SEQ ID NO: 1 of at least 60%,e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100%.

In another embodiment, the present invention relates to variants of themature polypeptide of SEQ ID NO: 2 comprising a substitution, deletion,and/or insertion at one or more (e.g., several) positions. In anembodiment, the number of amino acid substitutions, deletions and/orinsertions introduced into the mature polypeptide of SEQ ID NO: 2 is notmore than 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9. The amino acid changesmay be of a minor nature, that is conservative amino acid substitutionsor insertions that do not significantly affect the folding and/oractivity of the protein; small deletions, typically of 1-30 amino acids;small amino- or carboxyl-terminal extensions, such as an amino-terminalmethionine residue; a small linker peptide of up to 20-25 residues; or asmall extension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. Commonsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant mutantmolecules are tested for phospholipase C activity to identify amino acidresidues that are critical to the activity of the molecule. See also,Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site ofthe enzyme or other biological interaction can also be determined byphysical analysis of structure, as determined by such techniques asnuclear magnetic resonance, crystallography, electron diffraction, orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, for example, de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver etal., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acidscan also be inferred from an alignment with a related polypeptide.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

Sources of Polypeptides Having Phospholipase C Activity

A polypeptide having phospholipase C activity of the present inventionmay be obtained from microorganisms of any genus. For purposes of thepresent invention, the term “obtained from” as used herein in connectionwith a given source shall mean that the polypeptide encoded by apolynucleotide is produced by the source or by a strain in which thepolynucleotide from the source has been inserted. In one aspect, thepolypeptide obtained from a given source is secreted extracellularly.

The polypeptide may be a fungal polypeptide. For example, thepolypeptide may be a yeast polypeptide such as a Candida, Kluyveromyces,Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia polypeptide; ora filamentous fungal polypeptide such as an Acremonium, Agaricus,Alternaria, Aspergillus, Aureobasidium, Botryosphaeria, Ceriporiopsis,Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis,Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia,Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex,Kinochaeta, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus,Meripilus, Metarhizium, Mucor, Myceliophthora, Neocallimastix,Neurospora, Paecilomyces, Penicillium, Phanerochaete, Piromyces,Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor,Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia,Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, orXylaria polypeptide.

In another aspect, the polypeptide is a Saccharomyces carlsbergensis,Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomycesdouglasii, Saccharomyces kluyveri, Saccharomyces norbensis, orSaccharomyces oviformis polypeptide.

In another aspect, the polypeptide is an Acremonium cellulolyticus,Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus,Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Chrysosporium inops,Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporiummerdarium, Chrysosporium pannicola, Chrysosporium queenslandicum,Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsuiphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa,Irpex lacteus, Kinochaeta sp., Mucor miehei, Myceliophthora thermophila,Neurospora crassa, Metarhizium anisopliae, Penicillium emersonii,Aspergillus fumigates, Penicillium funiculosum, Penicillium oxalicum,Penicillium purpurogenum, Penicillium Phanerochaete chrysosporium,Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa,Thielavia australeinsis, Thielavia fimeti, Thielavia microspora,Thielavia ovispora, Thielavia peruviana, Thielavia setosa, Thielaviaspededonium, Thielavia subthermophila, Thielavia terrestris, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride polypeptide.

In a preferred aspect, the polypeptide is a Penicillium emersoniipolypeptide.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The polypeptide may be identified and obtained from other sourcesincluding microorganisms isolated from nature (e.g., soil, composts,water, etc.) or DNA samples obtained directly from natural materials(e.g., soil, composts, water, etc.) using the above-mentioned probes.Techniques for isolating microorganisms and DNA directly from naturalhabitats are well known in the art. A polynucleotide encoding thepolypeptide may then be obtained by similarly screening a genomic DNA orcDNA library of another microorganism or mixed DNA sample. Once apolynucleotide encoding a polypeptide has been detected with theprobe(s), the polynucleotide can be isolated or cloned by utilizingtechniques that are known to those of ordinary skill in the art (see,e.g., Sambrook et al., 1989, supra).

Polynucleotides

The present invention also relates to isolated polynucleotides encodinga polypeptide of the present invention, as described herein.

The techniques used to isolate or clone a polynucleotide are known inthe art and include isolation from genomic DNA or cDNA, or a combinationthereof. The cloning of the polynucleotides from genomic DNA can beeffected, e.g., by using the well-known polymerase chain reaction (PCR)or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligation activated transcription (LAT) andpolynucleotide-based amplification (NASBA) may be used. Thepolynucleotides may be cloned from a strain of Penicillium, or a relatedorganism and thus, for example, may be an allelic or species variant ofthe polypeptide encoding region of the polynucleotide.

Modification of a polynucleotide encoding a polypeptide of the presentinvention may be necessary for synthesizing polypeptides substantiallysimilar to the polypeptide. The term “substantially similar” to thepolypeptide refers to non-naturally occurring forms of the polypeptide.These polypeptides may differ in some engineered way from thepolypeptide isolated from its native source, e.g., variants that differin specific activity, thermostability, pH optimum, or the like. Thevariants may be constructed on the basis of the polynucleotide presentedas the mature polypeptide coding sequence of SEQ ID NO: 1, e.g., asubsequence thereof, and/or by introduction of nucleotide substitutionsthat do not result in a change in the amino acid sequence of thepolypeptide, but which correspond to the codon usage of the hostorganism intended for production of the enzyme, or by introduction ofnucleotide substitutions that may give rise to a different amino acidsequence. For a general description of nucleotide substitution, see,e.g., Ford et al., 1991, Protein Expression and Purification 2: 95-107.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide of the present invention operably linked to one or morecontrol sequences that direct the expression of the coding sequence in asuitable host cell under conditions compatible with the controlsequences.

A polynucleotide may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the polynucleotide priorto its insertion into a vector may be desirable or necessary dependingon the expression vector. The techniques for modifying polynucleotidesutilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter, a polynucleotide that isrecognized by a host cell for expression of a polynucleotide encoding apolypeptide of the present invention. The promoter containstranscriptional control sequences that mediate the expression of thepolypeptide. The promoter may be any polynucleotide that showstranscriptional activity in the host cell including mutant, truncated,and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xyIA and xyIB genes,Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994,Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trcpromoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicoloragarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroffet al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as thetac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in “Useful proteins fromrecombinant bacteria” in Gilbert et al., 1980, Scientific American 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promotersare disclosed in WO 99/43835.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Dania (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a modified promoter from an Aspergillus neutral alpha-amylasegene in which the untranslated leader has been replaced by anuntranslated leader from an Aspergillus triose phosphate isomerase gene;non-limiting examples include modified promoters from an Aspergillusniger neutral alpha-amylase gene in which the untranslated leader hasbeen replaced by an untranslated leader from an Aspergillus nidulans orAspergillus oryzae triose phosphate isomerase gene); and mutant,truncated, and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminator isoperably linked to the 3′-terminus of the polynucleotide encoding thepolypeptide. Any terminator that is functional in the host cell may beused in the present invention.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rrnB).

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans anthranilate synthase,Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase,Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-likeprotease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis cryIIIA gene (WO 94/25612) and a Bacillussubtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leader isoperably linked to the 5′-terminus of the polynucleotide encoding thepolypeptide. Any leader that is functional in the host cell may be used.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the polynucleotide and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus niger glucoamylase, Aspergillus nigeralpha-glucosidase Aspergillus oryzae TAKA amylase, and Fusariumoxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a polypeptide anddirects the polypeptide into the cell's secretory pathway. The 5′-end ofthe coding sequence of the polynucleotide may inherently contain asignal peptide coding sequence naturally linked in translation readingframe with the segment of the coding sequence that encodes thepolypeptide. Alternatively, the 5′-end of the coding sequence maycontain a signal peptide coding sequence that is foreign to the codingsequence. A foreign signal peptide coding sequence may be required wherethe coding sequence does not naturally contain a signal peptide codingsequence. Alternatively, a foreign signal peptide coding sequence maysimply replace the natural signal peptide coding sequence in order toenhance secretion of the polypeptide. However, any signal peptide codingsequence that directs the expressed polypeptide into the secretorypathway of a host cell may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a polypeptide. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of apolypeptide and the signal peptide sequence is positioned next to theN-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the polypeptide relative to the growth of the host cell.Examples of regulatory systems are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysystems in prokaryotic systems include the lac, tac, and trp operatorsystems. In yeast, the ADH2 system or GAL1 system may be used. Infilamentous fungi, the Aspergillus niger glucoamylase promoter,Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used. Other examples of regulatorysequences are those that allow for gene amplification. In eukaryoticsystems, these regulatory sequences include the dihydrofolate reductasegene that is amplified in the presence of methotrexate, and themetallothionein genes that are amplified with heavy metals. In thesecases, the polynucleotide encoding the polypeptide would be operablylinked with the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleotideand control sequences may be joined together to produce a recombinantexpression vector that may include one or more convenient restrictionsites to allow for insertion or substitution of the polynucleotideencoding the polypeptide at such sites. Alternatively, thepolynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the polynucleotide into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis dal genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spectinomycin, or tetracycline resistance. Suitable markers for yeasthost cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2,MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungalhost cell include, but are not limited to, amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell areAspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and aStreptomyces hygroscopicus bar gene.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAM111permitting replication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a polypeptide. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide of the present invention operably linked to one or morecontrol sequences that direct the production of a polypeptide of thepresent invention. A construct or vector comprising a polynucleotide isintroduced into a host cell so that the construct or vector ismaintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thepolypeptide and its source.

The host cell may be any cell useful in the recombinant production of apolypeptide of the present invention, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram-positive or Gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell including, butnot limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen.Genet. 168: 111-115), competent cell transformation (see, e.g., Youngand Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), orconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may be effectedby protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.166: 557-580) or electroporation (see, e.g., Dower et al., 1988, NucleicAcids Res. 16: 6127-6145). The introduction of DNA into a Streptomycescell may be effected by protoplast transformation, electroporation (see,e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405),conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl.Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may be effected by electroporation (see, e.g., Choi etal., 2006, J. Microbiol. Methods 64: 391-397) or conjugation (see, e.g.,Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). Theintroduction of DNA into a Streptococcus cell may be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804), or conjugation(see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, anymethod known in the art for introducing DNA into a host cell can beused.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell. The host cell may be a fungal cell. “Fungi” asused herein includes the phyla Ascomycota, Basidiomycota,Chytridiomycota, and Zygomycota as well as the Oomycota and allmitosporic fungi (as defined by Hawksworth et al., In, Ainsworth andBisby's Dictionary of The Fungi, 8th edition, 1995, CAB International,University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, Passmore, and Davenport,editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phiebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsuiphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phiebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a polypeptideof the present invention, comprising (a) cultivating a cell, which inits wild-type form produces the polypeptide, under conditions conducivefor production of the polypeptide; and (b) recovering the polypeptide.In a preferred aspect, the cell is a Penicillium cell. In a morepreferred aspect, the cell is a Penicillium emersonii cell.

The present invention also relates to methods of producing a polypeptideof the present invention, comprising (a) cultivating a recombinant hostcell of the present invention under conditions conducive for productionof the polypeptide; and (b) recovering the polypeptide.

The host cells are cultivated in a nutrient medium suitable forproduction of the polypeptide using methods known in the art. Forexample, the cell may be cultivated by shake flask cultivation, orsmall-scale or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors performed in a suitable medium and under conditions allowingthe polypeptide to be expressed and/or isolated. The cultivation takesplace in a suitable nutrient medium comprising carbon and nitrogensources and inorganic salts, using procedures known in the art. Suitablemedia are available from commercial suppliers or may be preparedaccording to published compositions (e.g., in catalogues of the AmericanType Culture Collection). If the polypeptide is secreted into thenutrient medium, the polypeptide can be recovered directly from themedium. If the polypeptide is not secreted, it can be recovered fromcell lysates.

The polypeptide may be detected using methods known in the art that arespecific for the polypeptides. These detection methods include, but arenot limited to, use of specific antibodies, formation of an enzymeproduct, or disappearance of an enzyme substrate. For example, an enzymeassay may be used to determine the activity of the polypeptide.

The polypeptide may be recovered using methods known in the art. Forexample, the polypeptide may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The polypeptide may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, VCH Publishers, New York, 1989) to obtain substantiallypure polypeptides.

In an alternative aspect, the polypeptide is not recovered, but rather ahost cell of the present invention expressing the polypeptide is used asa source of the polypeptide.

Plants

The present invention also relates to isolated plants, e.g., atransgenic plant, plant part, or plant cell, comprising a polynucleotideof the present invention so as to express and produce a polypeptide inrecoverable quantities. The polypeptide may be recovered from the plantor plant part. Alternatively, the plant or plant part containing thepolypeptide may be used as such for improving the quality of a food orfeed, e.g., improving nutritional value, palatability, and rheologicalproperties, or to destroy an antinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.Specific plant cell compartments, such as chloroplasts, apoplasts,mitochondria, vacuoles, peroxisomes and cytoplasm are also considered tobe a plant part. Furthermore, any plant cell, whatever the tissueorigin, is considered to be a plant part. Likewise, plant parts such asspecific tissues and cells isolated to facilitate the utilization of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seed coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing the polypeptide may beconstructed in accordance with methods known in the art. In short, theplant or plant cell is constructed by incorporating one or moreexpression constructs encoding the polypeptide into the plant hostgenome or chloroplast genome and propagating the resulting modifiedplant or plant cell into a transgenic plant or plant cell.

The present invention also relates to methods of producing a polypeptideof the present invention comprising (a) cultivating a transgenic plantor a plant cell comprising a polynucleotide encoding the polypeptideunder conditions conducive for production of the polypeptide; and (b)recovering the polypeptide.

Compositions

The present invention also relates to compositions comprising apolypeptide of the present invention. The composition may comprise apolypeptide of the present invention as the major enzymatic component,e.g., a mono-component composition. Alternatively, the composition maycomprise additional enzymes, such as an aminopeptidase, amylase,carbohydrase, carboxypeptidase, catalase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase,alpha-galactosidase, beta-galactosidase, glucoamylase,alpha-glucosidase, beta-glucosidase, haloperoxidase, invertase, laccase,lipase, mannosidase, oxidase, pectinolytic enzyme, peptidoglutaminase,peroxidase, phytase, polyphenoloxidase, proteolytic enzyme,ribonuclease, transglutaminase, or xylanase. The further enzyme may alsobe a polypeptide having phospholipase A1, A2, B and/or D activity. Theadditional enzyme(s) may be produced, for example, by a microorganismbelonging to the genus Aspergillus, e.g., Aspergillus aculeatus,Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus,Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, orAspergillus oryzae; Fusarium, e.g., Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium suiphureum, Fusariumtoruloseum, Fusarium trichothecioides, or Fusarium venenatum; Humicola,e.g., Humicola insolens or Humicola lanuginosa; or Trichoderma, e.g.,Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride.

The compositions may be prepared in accordance with methods known in theart and may be in the form of a liquid or a dry composition. Forinstance, the composition may be in the form of a granulate or amicrogranulate. The polypeptide may be stabilized in accordance withmethods known in the art.

Uses

The present invention is also directed to methods for using thepolypeptides having phospholipase C activity, or compositions thereof.

The phospholipase of the invention may be applied in a processcomprising treatment of a phospholipid or lysophospholipid with thephospholipase. Upon contacting with the phospholipase C the phospholipidor lysophospholipid is hydrolysed to yield diglyceride and a phosphateester, or monoglyceride and a phosphate ester, respectively.

The phospholipase of the invention may be applied in a processcomprising degumming of vegetable oil, e.g. an edible vegetable oil, ina process comprising hydrolysis of phospholipids to obtain improvedphospholipid emulsifiers, in particular wherein said phospholipid islecithin, in a process comprising hydrolysis of phospholipids in the gumfraction from water degumming to release entrapped triglyceride oil, ina process for improving the filterability of an aqueous solution orslurry of carbohydrate origin which contains phospholipid, and/or in aprocess for making a baked product, comprising adding the phospholipaseto a dough, and baking the dough to make the baked product.

A polypeptide of the present invention may be used for degumming anaqueous carbohydrate solution or slurry to improve its filterability,particularly, a starch hydrolysate, especially a wheat starchhydrolysate which is difficult to filter and yields cloudy filtrates.The treatment may be performed using methods well known in the art. See,for example, EP 219,269, EP 808,903.

A polypeptide of the present invention may be used in a process toreduce the phospholipid content in an edible oil. See, for example, WO2007/103005 and US 2008/0182322. Such a process is applicable to thepurification of any edible oil which contains phospholipid, e.g.,vegetable oil such as soybean oil, rape seed oil, and sunflower oil.

The phospholipase treatment can be carried out directly in the crude oilor after removal of slime (mucilage) e.g. by wet refining. After wetrefining the oil typically will contain 50-250 ppm of phosphorus asphospholipid at the beginning of the treatment with the phospholipase,and the treatment may reduce the phosphorus value, preferably to below11 ppm, such as to below 5-10 ppm.

The phospholipase treatment is conducted by dispersing an aqueoussolution of the phospholipase, preferably as droplets with an averagediameter below 10 microM. The amount of water is preferably 0.5-5% byweight in relation to the oil. An emulsifier may optionally be added.Mechanical agitation may be applied to maintain the emulsion. Thephospholipase treatment can be conducted at a pH in the range of about1.5 to about 7.0, preferably 3.5 to about 6. A suitable temperature isgenerally 30-70° C. (particularly 40-60° C., e.g., 55-55° C.).

The reaction time will typically be 1-12 hours (e.g., 1-6 hours, or 1-3hours). A suitable enzyme dosage will usually be 0.1-10 mg per liter(e.g., 0.5-5 mg per liter). The phospholipase treatment may be conductedbatchwise, e.g., in a tank with stirring, or it may be continuous, e.g.,a series of stirred tank reactors. The phospholipase treatment may befollowed by separation of an aqueous phase and an oil phase. Theseparation may be performed by conventional means, e.g., centrifugation.When a liquid lipase is used the aqueous phase will containphospholipase, and the enzyme may be re-used to improve the processeconomy.

A polypeptide of the present invention and other such polypeptideshaving activity towards one or more of the four major phospholipidsphosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidicacid (PA) and phosphatidyl inositol (PI) may be used for degumming anoil composition. Accordingly the invention provides a method fordegumming an oil composition, the method comprising (a) providing an oilcomposition containing a quantity of phospholipids, (b) contacting saidoil composition with a phospholipase C enzyme under conditionssufficient for the enzyme to react with the phospholipids to creatediacylglycerol and phosphate ester, and, (c) separating the phosphateester from the oil composition, thereby obtaining a degummed oilcomposition, wherein the phospholipase C enzyme has activity towards oneor more of phosphatidylcholine (PC), phosphatidylethanolamine (PE),phosphatidic acid (PA) and phosphatidyl inositol (PI).

In addition to the phospholipase C of the present invention a furtherenzyme may be applied in the degumming process outlined above. In apreferred embodiment the further enzyme is a polypeptide havingphospholipase A1, A2, B and/or D activity. A suitable polypeptide havingphospholipase A1 activity may be Lecitase Ultra available from NovozymesA/S. A suitable polypeptide having phospholipase D activity may be e.g.,an enzyme derived from Saccharomyces cerevisiae and having the sequenceUniProt: P36126, or an enzyme derived from Dictyostelium discoideum andhaving the sequence UniProt: Q54Z25.

The degumming process may comprise (a) providing an oil compositioncontaining a quantity of PC, PE, and/or PI, (b) treating said oilcomposition with a phospholipase D enzyme to convert PC, PE and/or PI,into PA, (c) treating said oil composition with a phospholipase C enzymeto convert PA in to diglyceride and phosphoric acid. The phospholipase Dand phospholipase C may be applied together such that steps (b) and (c)occur substantially simultaneously.

Immobilization of the phospholipase on a suitable carrier may also beapplied using any method known in the art incl. by entrapment in naturalor synthetic matrices, such as hydrophobic polymers, ion exchangedresins, sol-gels, alginate, and carrageenan; by cross-linking methodssuch as in cross-linked enzyme crystals (CLEC) and cross-linked enzymeaggregates (CLEA); or by precipitation on salt crystals such asprotein-coated micro-crystals (PCMC).

In certain embodiments the present invention relates to a method ofproducing a fatty acid ester product, wherein the carrier is ahydrophilic carrier selected from the group containing: porousin-organic particles composed of alumina, silica and silicates such asporous glas, zeolites, diatomaceous earth, bentonite, vermiculite,hydrotalcite; and porous organic particles composed of carbohydratepolymers such as agarose or cellulose. In other embodiments the presentinvention relates to a method of producing a fatty acid ester product,wherein the carrier is a hydrophobic polymeric carrier, e.g.polypropylen, polyethylene, acrylate. Suitable commercial carriers aree.g. LEWATIT™, ACCUREL™, PUROLITE™ and AMBERLITE™.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES Example 1

Phospholipase C activity assay: Reaction mixtures comprising 10 microLof a 100 mM p-nitrophenyl phosphoryl choline (p-NPPC) solution in 100 mMBorax-HCl buffer, pH 7.5 and 90 microL of the enzyme solution are mixedin a microtiter plate well at ambient temperature. The microtiter plateis then placed in a microtiter plate reader and the releasedp-nitrophenol is quantified by measurement of absorbance at 410 nm.Measurements are recorded during 30 min at one minute intervals.Calibration curves in the range 0.01-1 microL/ml p-nitrophenol areprepared by diluting a 10 micromol/ml p-nitrophenol stock solution fromSigma in Borax-HCl buffer. One unit will liberate 1.0 micromol/minute ofp-NPPC at ambient temperature.

Example 2

The thermostability of the phospholipase C of the present invention(EmPLC5) was determined at pH 4.0, pH 5.5 and pH 7.0 by DifferentialScanning calorimetry (DSC) using a VP-Capillary Differential Scanningcalorimeter (MicroCal Inc., Piscataway, N.J., USA) at a proteinconcentration of approximately 0.5 mg/ml. The thermal denaturationtemperature, Td (° C.), was taken as the top of denaturation peak (majorendothermic peak) in thermograms (Cp vs. T) obtained after heatingenzyme solutions in buffer at a constant programmed heating rate of 200K/hr. Sample- and reference-solutions (approx. 0.2 ml) were loaded intothe calorimeter (reference: buffer without enzyme) from storageconditions at 10° C. and thermally pre-equilibrated for 20 minutes at20° C. prior to DSC scan from 20° C. to 100° C. Denaturationtemperatures (Td) were determined at an accuracy of approximately +/−1°C. (Table 1).

TABLE 1 Denaturation temperatures for EmPLC5 pH Buffer Td (° C.) 4.0Acetate 90 5.5 Acetate 88 7.0 HEPES 83

Example 3

Substrate Specificity

To ensure a uniform substrate with a high concentration of all fourrelevant phospholipids (PC, PE, PI and PA) a test substrate is producedfrom soy bean oil spiked with commercial lecithin. This substrate isthen incubed with enzyme and citrate buffer. Citrate is used since somedegumming applications involve preconditioning the oil with citric acid.After the desired reaction time, the reaction mixture is analyzed byP-NMR. This involves an aqueous extraction step during which thephosphor species liberated by the PLC are removed from the oil phase.Hence, only lipophilic P-species are detected, including unreactedphospholipid.

The substrate is a fully refined soybean oil with added lecithin (a 2:1ratio of the Sigma products P6636 (PI, CAS 97281-52-2) and 61755 (PC,CAS 8002-43-5)) added to a concentration of 50 mg/mL oil. This mixturewill also to contain PA and PE. Disperse 250 μL into 2 mL Eppendorftubes.

The assay is conducted at three different pH values, 4.0, 5.5 and 7.0 in100 mM Na-citrate buffer and an enzyme concentration of 0.9 mg/mL. Theenzyme amount applied is 100 mg EP/kg oil.

Before NMR analysis, samples are extracted with 100 mL 0.2 M Cs-EDTA pH7.5 solution. Preparation of this solution: EDTA (5.85 g) is dispersedin water (approx. 50 mL). The pH is adjusted to 7.5 using 50% w/w CsOH(approx. 30 mL), which will dissolve the EDTA completely. Water is addedup to 100 mL to give a concentration of 0.2 M EDTA.

Also, an internal standard solution (IS) of 2 mg/mL triphenylphosphate(TPP) in MeOH is used.

To the Eppendorf with 250 μL substrate in oil is added 25 μL dilutedenzyme. The Eppendorf is incubated in thermoshaker at 50° C. for 2 h,followed by NMR analysis as described below.

To the oil sample is then added 0.500 mL IS-solution, followed by 0.5 mLCDCl₃ and 0.5 mL Cs-EDTA buffer. Shake for 30 s, then do centrifugation(tabletop centrifuge, 1 min, 13,400 rpm) to get phase separation. Thelower phase is transferred to a NMR-tube.

P-NMR is performed with 128 scans and a delay time of 5 s. Scalereference is set according to IS signal (−17.75 ppm). Integrate allsignals. Assignments (approx. ppm @ 25 C): 1.7 (PA), −0.1 (PE), −0.5(PI), −0.8 (PC). The position of the signals can change significantlyaccording to exact pH value, temperature, sample concentration, etc. Theconcentration of each species is calculated as “ppm P”, i.e. mgelemental P per kg oil sample. Hence, ppm P=I/I(IS)*n(IS)*M(P)/m(oil).

The phospholipase of the invention EmPLC5 and the commercialphospholipase C Purifine were incubated with the test substrate andanalysed as described above. The results are shown below.

TABLE 2 Remaining (non-hydrolyzed) phospholipids in ppm P PA PE PI PCBlank 180 349 595 431 EmPLC5, pH 4.0 91 173 0 43 EmPLC5, pH 5.5 69 164 051 EmPLC5, pH 7.0 0 201 0 69 Purifine, pH 4.0 256 388 609 391 Purifine,pH 5.5 175 204 598 111 Purifine, pH 7.0 169 0 594 0

The data shows that the phospholipase C of the present invention(EmPLC5) has activity on all four phospholipids over a broad pH range.In contrast, Purifine has activity on PE and PC only at neutral pH.

Example 4

Performance in Degumming Assay

Performance/activity of the P. emersonii phospholipase (EmPLC5) indegumming application was determined in an assay (described below) thatmimics industrial scale degumming, followed by oil phase measurement ofa) diglyceride content by High-performance liquid chromatography (HPLC)coupled to Evaporative Light Scattering Detector (ELSD), and b)quantification of the individual phospholipids species:Phosphatidylcholine (PC); Phosphatidylinositol (PI);Phosphatidylethanolamine (PE); Phosphatidic acid (PA);Phosphatidylserine (PS) by HPLC-mass spectrometry (MS) and C) totalphosphorous reduction by Inductively coupled plasma optical emissionspectrometry (ICP-OES).

Degumming Assay

Crude soybean oil (25-75 g) was initially acid/base pretreated (or not)to facilitate conversion of insoluble phospholipids salt into morehydratable forms and ensure an environment suitable for the enzyme.Acid/base pretreatment was done by acid addition and mixing inultrasonic bath (BRANSON 3510) for 5 min and incubation in rotator for15 min followed by base neutralization in ultrasonic bath for 5 min.Enzyme reaction was conducted in low aqueous system (3% water totalbased on oil amount) in centrifuge tubes. Samples were ultrasonictreated for 5 min, followed by mixing (Stuart SB3 rotator) underincubation in heated cabinet at selected temperature. Enzyme treatedoil+enzyme+water mix was then heated/centrifuged at 85° C., 15 min, 700g (Koehler Instruments, K600X2 oil centrifuge) to separate in an oilphase and a heavy water/gum phase.

Diglyceride Measurement

The HPLC-ELSD method (using DIONEX equipment and Lichrocart Si-60, 5 μm,Lichrosphere 250-4 mm, MERCK column) is based on the principle of theAOCS Official Method Cd 11d-96 and quantifies the diglyceride contentdown to 0.1 wt %.

Phosphorous/Phosphorlipid Measurement

The ICP-OES quantifies the phosphorous (P) content and other metals suchas Ca, Mg, Zn down to 4 ppm with an accuracy of approximately ±1 ppm P.

Example 5

The phospholipase of the invention was applied in a degumming experimentconducted over 24 hrs at various temperatures applying crude soybean oilspiked with 1% PI/PA lecithin. The treatments are shown in table 3 andthe results in table 4.

TABLE 3 The treatments comprised oil, phosphoric acid solution, sodiumhydroxide, water, enzyme and reaction time according to the schemebelow. Degumming with acid/base pretreatment Water degumming SampleBlank EmPLC5 Blank EmPLC5 Oil amount (g) 25 25 25 25 Phosphoric acid 8.78.7 0 0 85% (ul) 4M NaOH (ul) 25.5 25.5 0 0

TABLE 4 Diglyceride formed over time while degumming with phospholipaseEmPLC5 (diglyceride increase compared to blank sample). Acid/basepretreated oil prior to degumming Water degummed Temperature Reactiontime (hours) Reaction time (hours) (° C.) 2 4 24 2 4 24 55 0.00 0.471.17 0.00 0.62 1.09 65 0.00 0.02 0.28 0.27 0.35 0.77 70 0.29 0.43 1.500.26 0.48 1.55 75 0.39 0.59 1.34 0.46 0.65 1.54 80 0.21 0.42 1.26 0.260.45 1.41

Degumming with the phospholipase C of the invention resulted insignificant diglyceride formation in a broad temperature range from 55°C. to 80° C., and the EmPLC5 appears very suitable for use at elevatedtemperatures of 70-80° C. Good performance in water degumming as well asacid assisted degumming is observed demonstrating robustness towardsvarying pH conditions.

Example 6

The phospholipase C EmPLC5 was used in degumming of a crude soybean oilrich in non-hydratable phospholipids (145 ppm P non-hydratablephospholipids out of 700 ppm P total). Degumming was performed at an oilamount of 75 g, at 70° C. for 24 hrs using varying levels of acidity (0,0.5, 1.0 and 1.5 eqv NaOH).

TABLE 5 Diglyceride formed during degumming with acid/base treatmentusing 0.05% phosphoric acid and varying equivalents of NaOH. Diglyceride% analysed by HPLC Diglyceride % Sample 0 hrs 2 hrs 4 hrs 24 hrs Blank 0eqv. NaOH 0.05% PA 0.43 0.54 0.43 0.44 Blank 0.5 eqv. NaOH 0.05% PA +0.5 eqv NaOH 0.43 0.42 0.43 0.47 Blank 1.0 eqv. NaOH 0.05% PA + 1.0 eqvNaOH 0.43 0.43 0.43 0.48 Blank 1.5 eqv. NaOH 0.05% PA + 1.5 eqv NaOH0.43 0.44 0.44 0.48 EmPLC5 0 eqv. NaOH 0.05% PA 0.43 0.55 0.50 0.88EmPLC5 0.5 eqv. NaOH 0.05% PA + 0.5 eqv NaOH 0.43 0.54 0.68 1.24 EmPLC51.0 eqv. NaOH 0.05% PA + 1.0 eqv NaOH 0.43 0.56 0.71 1.19 EmPLC5 1.5eqv. NaOH 0.05% PA + 1.5 eqv NaOH 0.43 0.44 0.46 0.76

TABLE 6 Free fatty acid and metals in degummed oil after degumming for24 h. Metal composition Sample FFA wt % P Ca Mg Zn Blank 0 eqv. NaOH1.12 99 55 19 17 Blank 0.5 eqv. NaOH 1.09 75 46 15 14 Blank 1.0 eqv.NaOH 1.04 101 70 26 18 Blank 1.5 eqv. NaOH 1.06 111 80 34 13 EmPLC5 0eqv. NaOH 1.11 52 58 16 12 EmPLC5 0.5 eqv. NaOH 1.07 36 70 20 12 EmPLC51.0 eqv. NaOH 1.07 52 50 14 8 EmPLC5 1.5 eqv. NaOH 1.09 138 123 46 14

The phospholipase of the invention EmPLC5 demonstrated high and robustperformance in acid assisted degumming with preference for an acidicdegumming environment only partly neutralized with sodium hydroxide. At0.05% phosphoric acid+partial neutralization with 0.5 equivalent NaOH(compared to acid dosage) significant diglyceride increase as well asphosphorous reduction was observed.

No free fatty acid increase was observed in the degumming assay whichdemonstrates that EmPLC5 is active on the phosphatide of thephospholipid molecule only.

Example 7

Based on the amino acid sequence in SEQ ID NO:2 a variant EmPLC5.1comprising the substitutions K57A, C173A, K180N, 1216V, V3201, D321E,T365L, 1397E, Q399H, Q518K and A558S was prepared. Preliminary datashowed that the stability of the variant was substantially unchanged andthe phospholipase C activity was maintained.

Example 8

The wild-type phospholipase EmPLC5 and the variant EmPLC5.1 applied indegumming. The diglyceride content and total content of phosphorousafter enzymatic degumming with/without acid/base pretreatment for 24hours were measured. The treatments are shown in table 7 and the resultsin table 8.

TABLE 7 The treatments comprised oil, phosphoric acid solution, sodiumhydroxide, water, enzyme and reaction time and temperature according tothe scheme below. Degumming with acid/base pretreatment Water degummingVariant Variant Sample Blank EmPLC5 EmPLC5.1 Blank EmPLC5 EmPLC5.1 Oilamount (g) 75.0 75.0 75.0 75.0 75.0 75.0 Phosphoric 26 26 26 — — — acid85% (ul) 4M NaOH (ul) 48 48 48 — — —

TABLE 8 Increase of diglyceride after enzyme treatment of acid/basepretreated soybean oil measured by HPLC-ELSD and content of totalphosphorous measured by ICP-OES. Crude soybean oil contains 711 mg/kg ofphosphorous Phosphorous content Sample Diglyceride increase (wt %) total(mg/kg) Reaction time (hours) 2 4 24 — EmPLC5 0.09 0.03 0.82 13 VariantEmPLC5.1 0.09 0.05 0.39 19

In the degumming assay the phospholipase C EmPLC5 as well as the variantEmPLC5.1 convert phospholipids into diglycerides. Both enzymes work wellin the acidic environment when oil is acid/base pre-treated.

TABLE 3C Content of individual phospholipids (PC, PE, PI, PA) afterenzymatic degumming for 24 hours of acid/base pretreated soy bean oil.Measured by HPLC-MS. PA PC PE PI mg/kg mg/kg mg/kg mg/kg EmPLC5 2.3 <2<2 1.5 Variant EmPLC5.1 4.7 <2 2.9 1.7

In acid assisted degumming conducted the phospholipase of the inventionEmPLC5 and the variant EmPLC5.1 reduces all phospholipid species (PA,PI, PC, PE).

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

The invention claimed is:
 1. A method for reducing the content ofphosphorus containing components in an edible oil, the methodcomprising: (a) contacting the oil with an aqueous solution of apolypeptide having phospholipase C activity, wherein the aqueoussolution is emulsified in the oil until the phosphorus content of theoil is reduced, and wherein the polypeptide has at least 90% sequenceidentity to the mature polypeptide of SEQ ID NO: 2; and (b) thenseparating the aqueous phase from the treated oil.
 2. The method ofclaim 1, wherein the polypeptide having phospholipase C activity has atleast 95% sequence identity to the mature polypeptide of SEQ ID NO: 2.3. The method of claim 1, wherein the polypeptide having phospholipase Cactivity has at least 97% sequence identity to the mature polypeptide ofSEQ ID NO:
 2. 4. The method of claim 1, wherein the polypeptide havingphospholipase C activity has at least 98% sequence identity to themature polypeptide of SEQ ID NO:
 2. 5. The method of claim 1, whereinthe polypeptide having phospholipase C activity has at least 99%sequence identity to the mature polypeptide of SEQ ID NO:
 2. 6. Themethod of claim 1, wherein the polypeptide having phospholipase Cactivity comprises the sequence of amino acid residues 1-594 of SEQ IDNO:
 2. 7. The method of claim 1, wherein the polypeptide havingphospholipase C activity is a fragment of the sequence of amino acidresidues 1-594 of SEQ ID NO:
 2. 8. The method of claim 1, wherein thepolypeptide is a variant of the mature polypeptide of SEQ ID NO: 2comprising a substitution, deletion, and/or insertion at one or morepositions.
 9. The method of claim 1, wherein the edible oil is avegetable oil.